Volume 25, Issue 10, Pages 2775-2783.e3 (December 2018) B Cells Produce the Tissue-Protective Protein RELMα during Helminth Infection, which Inhibits IL-17 Expression and Limits Emphysema  Fei Chen, Wenhui Wu, Lianhua Jin, Ariel Millman, Mark Palma, Darine W. El-Naccache, Katherine E. Lothstein, Chen Dong, Karen L. Edelblum, William C. Gause  Cell Reports  Volume 25, Issue 10, Pages 2775-2783.e3 (December 2018) DOI: 10.1016/j.celrep.2018.11.038 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 25, 2775-2783.e3DOI: (10.1016/j.celrep.2018.11.038) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Emphysematous Pathology Develops by 7 Days after Nb Inoculation, and Its Severity Is Reduced in Il17a−/− and Tcrd−/− Mice (A) Emphysematous pathology was determined by digital imaging analysis of mean linear intercept measurements (Lm) of alveolar spaces at days 2, 4, and 7 after N. brasiliensis (Nb) inoculation of WT BALB/c mice. (B–E) Il17a−/− and WT BL/6 control mice or Tcrd−/− and WT BL/6 control mice were inoculated with Nb. Then 8 days later, emphysematous pathology was assessed by Lm (B and D) and the total number of CD11b+Ly6G+ neutrophils at day 2 was determined by flow cytometry (C and E). Each symbol represents an individual mouse, and horizontal lines indicate the mean. Data shown are the mean and SEM from at least five individual mice per group and are representative of at least two independent experiments (∗∗p < 0.01). See also Figure S1. Cell Reports 2018 25, 2775-2783.e3DOI: (10.1016/j.celrep.2018.11.038) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 B Cell Deficiency Exacerbates Emphysematous Pathology after Nb Inoculation (A–E) BALB/c WT and Jh−/− mice were inoculated with 600 L3 and, 7 days later, were analyzed for emphysematous pathology and IL-17 expression. (A) Left lobe of lung was collected and photographed (scale bar, 5 mm). (B) H&E staining of representative formalin-fixed lung sections of mice. (C) Digital imaging analysis of mean linear intercept measurements (Lm) of alveolar spaces. (D) Number of lung IL-17A+ γδ T cells. (E) Number of neutrophils (CD11b+Ly6G+), analyzed by FACS. (F–I) WT and Jh−/− mice were inoculated with 300 L3 and, 30 days later, were analyzed for emphysematous pathology. H&E staining (F) and Lm analyses (G) were assessed as described earlier, and the number of lung IL-17A+ γδ T cells (H) and lung neutrophils (CD11b+Ly6G+) (I) was determined by flow cytometry. Each symbol represents an individual mouse, and horizontal lines indicate the mean. Data shown are representative of at least two independent experiments (∗p < 0.05, ∗∗p < 0.01). Cell Reports 2018 25, 2775-2783.e3DOI: (10.1016/j.celrep.2018.11.038) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Transfer of WT B Cells or Administration of Neutralizing Anti-IL-17A Antibody Restored Control of Emphysematous Pathology in B Cell-Deficient Nb-Inoculated Mice (A–D) BALB/c WT, B cell-deficient Jh−/−, and Jh−/− recipient mice with transferred BABL/c WT B cells were all assessed for severity of emphysematous pathology at day 7 and for neutrophils and Il17 gene expression at day 2 after Nb inoculation. (A) H&E staining of representative sections is shown. (B) Emphysematous pathology was measured using digital imaging analysis of mean linear intercept measurements (Lm) of alveolar spaces. (C) The number of lung neutrophils (CD11b+Ly6G+) was determined by flow cytometry at day 2. (D) Whole-lung Il17a gene expression by qPCR is presented as the fold increase over naive WT mice after normalization to 18s ribosomal RNA (18srRNA). (E and F) Jh−/− mice were administered blocking anti-IL-17A Ab and inoculated with Nb. Then 7 days later, they were assessed for emphysematous pathology through Lm analysis (E), and 2 days later, they were analyzed for number of lung neutrophils (F); results are presented as described earlier. Each symbol represents an individual mouse. Horizontal lines indicate the mean (B, C, E, and F), or data shown are the mean and SEM from at least five individual mice per group (D). Data shown are representative of at least two independent experiments (∗p < 0.05, ∗∗p < 0.01). See also Figure S2. Cell Reports 2018 25, 2775-2783.e3DOI: (10.1016/j.celrep.2018.11.038) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 B Cell Deficiency Did Not Exacerbate Acute Lung Injury WT and Jh−/− mice were inoculated with Nb and analyzed for acute lung injury (ALI) and type 2 immunity at day 4. (A–C) BAL red blood cell (RBC) count (A), ALI pathology scores (B), and BAL eosinophil count (C). (D–F) Lung tissues were examined for the expression of Il13 (D), Arginase 1 (Arg1) (E), and IGF1 mRNA (F) by qPCR. Gene expression is presented as the fold increase over naive WT mice after normalization to 18sRNA. Each symbol represents an individual mouse. Horizontal lines indicate the mean (A and C), or data shown are the mean and SEM from five individual mice per group (B–F). Data shown are representative of at least two independent experiments (∗∗p < 0.01). Cell Reports 2018 25, 2775-2783.e3DOI: (10.1016/j.celrep.2018.11.038) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 IL-4R Signaling Promotes B Cell-Mediated Control of Emphysema B cells from WT and Il4ra−/− mice were transferred to Jh−/− recipients at days −3, 0, and +1 during N. brasiliensis inoculation. Lungs were collected for analysis 7 days after inoculation. (A) H&E staining of formalin-fixed lung sections. (B) Emphysematous pathology was quantitated by digital imaging analysis of mean linear intercept measurements (Lm) of alveolar spaces. (C) Number of lung neutrophils (CD11b+Ly6G+) was determined by flow cytometry. Each symbol represents an individual mouse, and horizontal lines indicate the mean. (D) Lung tissues were examined for the expression of Il17a by qPCR. Gene expression is presented as the fold increase over naive WT mice after normalization to 18sRNA and expressed as the mean and SEM from five individual mice per group. Data shown are representative of at least two independent experiments (∗p < 0.05, ∗∗p < 0.01). See also Figure S3. Cell Reports 2018 25, 2775-2783.e3DOI: (10.1016/j.celrep.2018.11.038) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 RELMα Expression by B Cells Is Required for Limiting Nb-Induced Emphysematous Pathology (A) B cell numbers in the lung at different time points after N. brasiliensis inoculation were assessed by flow cytometry. (B) At day 5 after Nb inoculation, sort-purified B cells were analyzed for mRNA expression of candidate regulatory factors. (C) Sort-purified B cells from Il4ra−/− and WT BALB/c mice were analyzed for Retnla mRNA expression at day 5 after Nb inoculation. (D–F) Sort-purified B cells (D), alveolar macrophages (E), or non-alveolar macrophages (F) at different time points after Nb infection were analyzed for Retnla mRNA expression. (G and H) RELMα mean fluorescence intensity (MFI) of lung B cells (CD19+) (G) and alveolar macrophages (H) was determined by cytoplasmic staining and FACS at day 5 after N. brasiliensis inoculation. (I and J) Sort-purified B cells from either untreated Retnla−/− or WT mice were transferred to recipient Jh−/− mice at days −3, 0, and +1 after N. brasiliensis inoculation. Lungs were collected for analysis 7 days after inoculation, emphysematous pathology was digitally imaged as described in Figure 1 (I), and lung tissues were analyzed for the expression of Il17a by qPCR (J). (K) Retnla−/− and WT mice were inoculated with Nb, and 2 days later, γδ T cells were assessed for IL-17A production by intracellular staining and flow cytometric analyses. (L and M) FACS analysis of lung B cell subsets at day 5 after N. brasiliensis inoculation, showing expression of IgM and IgD (L) and CD21, CD23, and CD24 (M) on gated CD19+ lymphocytes. Each symbol represents an individual mouse. Small horizontal lines indicate the mean (A and D–I), or data shown are the mean and SEM from five individual mice per group (B–F and J) or a pool of five mice per group (K and L). Data shown are representative of at least two independent experiments (∗∗p < 0.01). See also Figures S4 and S5. Cell Reports 2018 25, 2775-2783.e3DOI: (10.1016/j.celrep.2018.11.038) Copyright © 2018 The Author(s) Terms and Conditions